MALDI-TOF Mass Spectrometry Applied to Identifying Species of Insect-Pathogenic Fungi from the Metarhizium Anisopliae Complex

Mycologia

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MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex

Rogério Biaggioni Lopes, Marcos Faria, Daniela Aguiar Souza, Carlos Bloch Jr., Luciano P. Silva & Richard A. Humber

To cite this article: Rogério Biaggioni Lopes, Marcos Faria, Daniela Aguiar Souza, Carlos Bloch Jr., Luciano P. Silva & Richard A. Humber (2014) MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhiziumanisopliae complex, Mycologia, 106:4, 865-878, DOI: 10.3852/13-401 To link to this article: https://doi.org/10.3852/13-401

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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=umyc20 Mycologia, 106(4), 2014, pp. 865–878. DOI: 10.3852/13-401 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex

Roge´rio Biaggioni Lopes pingshaense-anisopliae-robertsii-brunneum clade). Due Marcos Faria to the increasing frequency of phylogenetically Daniela Aguiar Souza (genomically) based taxonomic revisions of fungi, Carlos Bloch Jr. this approach is especially useful for culture collec- Luciano P. Silva tions, because once the protein profiles of Metarhi- Embrapa Genetic Resources and Biotechnology, Brasilia zium isolates are obtained taxonomic updating of DF, CP 02372, 70770-917, Brazil MALDI-TOF library data is easily accomplished by Richard A. Humber1 comparing stored profiles with those of newly USDA-ARS Biological Integrated Pest Management proposed taxa. Research, Robert W. Holley Center for Agriculture and Key words: Ascomycota, biocontrol, Clavicipita- Health, 538 Tower Road, Ithaca, New York 14853 ceae, insect fungi, methodology, mycoinsecticides, phylogeny

Abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) INTRODUCTION has proven to be a powerful tool for taxonomic Classical taxonomy based on morphological charac- resolution of microorganisms. In this proof-of-con- ters is now widely understood to pose a series of cept study, we assessed the effectiveness of this challenges, including poor resolution and variability technique to track the current gene sequence-based of traits, also requiring relatively lengthy identifica- phylogenetic classification of species in the Metarhi- tion processes (Santos et al. 2010). The taxonomic zium anisopliae complex. Initially the phylogenetic classification or identification of fungi has undergone analysis of 51 strains by sequencing of the 59 end of major advances in the past decades, mainly following the TEF-1a gene region revealed seven species within the adoption of molecular approaches (Hibbett et al. M. anisopliae sensu lato and two varieties outside this 2007). Phylogenetically based species complexes have complex. Because initial studies on MS profiles from been resolved and taxonomic revisions proposed for a different cell types showed that mycelial fragments or number of insect fungal pathogens (Zare and Gams conidia produced on nutrient-poor medium may 2001, Zare et al. 2001, Luangsa-ard et al. 2004, Rehner yield too much background noise, all subsequent and Buckley 2005, Sung et al. 2007, Bischoff et al. spectrometric analyses were performed with acid- 2009, Rehner et al. 2011). Works focusing on multi- hydrolyzed conidia from 10–12 d old PDA cultures. locus phylogenetic analyses have clarified phyloge- The initial MALDI-TOF reference library included netic relationships within the hypocrealean genera protein spectral profiles from nine taxonomically Metarhizium (Bischoff et al. 2009) and Beauveria distinct, molecularly identified isolates sharing high (Rehner et al. 2011) and yielded well justified genetic homology with the ex-type or ex-epitype proposals of new taxa within species complexes. isolates of these taxa in Metarhizium.Asecond Microbial culture collections are valuable sources of reference library added one isolate each for M. authenticated germplasm for basic and applied anisopliae sensu stricto and M. robertsii. The second, research, for studies on biodiversity and systematics larger reference library (including 11 taxa) allowed and also may serve as secure repositories for nearly perfect MALDI-TOF matching of DNA-based industrially important strains (Santos and Lima species identification for the 40 remaining isolates 2001, Smith 2003). Government culture collections, molecularly recognized as M. anisopliae sensu stricto such as the USDA-ARS Collection of Entomopatho- (n 5 19), M. robertsii (n 5 6), M. majus (n 5 3), M. genic Fungal Cultures (ARSEF; Ithaca, New York) and lepidiotae (n 5 1), M. acridum (n 5 3), M. flavoviride the Invertebrate Fungal Collection at Embrapa var. pemphigi (n 5 1), plus seven unidentified strains Genetic Resources and Biotechnology (Brasilia DF, (six of them phylogenetically close to M. anisopliae Brazil), hold large numbers of insect fungi from sensu stricto and one outside the Metarhizium economically important species complexes, whose taxa show few or no clear morphological differences. Submitted 24 Dec 2013; accepted for publication 5 Feb 2014. Such collections are challenged to assure that their 1 Corresponding author. E-mail: [email protected] catalogs reflect current, well justified classifications

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Published online 20 Jan 2017 866 MYCOLOGIA that incorporate ongoing phylogenetically based clade (M. pingshaense-anisopliae-robertsii-brunneum) within taxonomic revisions. While almost all current molec- the M. anisopliae species complex (Lopes et al. 2013b), was ularly based changes derive from gene sequence included as a further test of the robustness of MALDI-TOF comparisons, alternative molecular profile technolo- profiles in comparison to sequence-based analyses. gies, such as matrix-assisted laser desorption/ioniza- Strains were inoculated on potato dextrose agar (PDA; Acumedia-Neogen Corp., Lansing, Michigan, and main- tion time-of-flight mass spectrometry (MALDI-TOF tained at 25 6 0.5 C and 12 h of photophase. Five-day-old MS), can allow low-cost, rapid and reliable identifica- culture plugs (8 mm diam) were inoculated into 500 mL tion for a wide range of microorganisms (Santos et al. Erlenmeyer flasks containing 250 mL Sabouraud dextrose 2010, Croxatto et al. 2012) including bacteria (Anhalt yeast broth (SDY broth; 1% glucose, 0.3% malt extract, 0.5% and Fenselau 1975, Carbonnelle et al. 2011), yeasts peptone, 0.3% yeast extract), followed by a 4 D incubation (Qian et al. 2008, Marklein et al. 2009, Bader et al. at 25 6 0.5 C in a rotary shaker at 250 rpm. Mycelial samples 2011, Pinto et al. 2011) and such taxonomically were harvested on filter paper by vacuum filtration, frozen difficult genera of filamentous fungi as Fusarium at 270 C, and then lyophilized and stored at 220 C until (Seyfarth et al. 2008, Dong et al. 2009, Kemptner et al. use. For MALDI-TOF studies, conidial samples (4 mg) were 2009, Marinach-Patrice et al. 2009), Aspergillus (Li et gently collected from 10–12 d old cultures with a glass rod, al. 2000, Hettick et al. 2008, Rodrigues et al. 2011), avoiding fragments of mycelia and culture medium, and immediately transferred to 2 mL Eppendorf tubes contain- Penicillium (Welham et al. 2000, Chen and Chen ing 1 mL 75% ethanol. The suspension was vortexed for 30 s 2005) and Trichoderma (de Respinis et al. 2010, and centrifuged for 2 min at 16 100 3 g. The supernatant Samuels et al. 2010). However, this approach has not was discarded, and pellets were air-dried at room temper- been applied to insect fungi until now. ature for 15 min and maintained at 212 C until use. The availability of practical and reliable identification techniques that also would allow rapid reclassi- Molecular identification of Metarhizium strains.—Molecu- fication without relatively expensive and time con- lar studies were performed to select strains sharing high homology with ex-type/ex-epitype isolates mentioned by suming (PCR-based) re-analyses potentially could Bischoff et al. (2009), so these strains could be further used improve the quality of taxonomic information pro- for creation of main spectrum profiles (MSPs) in the vided by fungal culture collections worldwide, includ- MALDI-TOF MS studies. For each strain, approximately ing those concentrated on insect fungi. Therefore, 25 mg frozen hyphae were crushed in a mortar under liquid the present proof-of-concept study seeks to test nitrogen and the total genomic DNA was extracted and whether MALDI-TOF MS can be adjusted for the processed further as described by Lopes et al. (2013a). reliable tracking of gene sequence-based identifica- Phylogenetic identification was performed by amplifying tion within the economically important and globally the 59 end of TEF-1a (translation elongation factor 1a) distributed Metarhizium anisopliae species complex. gene fragment. Sequencing was performed by Macrogen Inc. (Seoul, South Korea) and edited with DNA baser (DNABaser Sequence Assembler 3, Heracle Biosoft, Pitesti, MATERIALS AND METHODS Romania). Consensus sequences were analyzed with MEGA 5.03 software (Tamura et al. 2011), and the final alignments Source of fungi and their cultivation.—Fifty-one strains from were adjusted manually. Selected TEF-1a sequences from the genus Metarhizium were used. Isolates with code CG (n GenBank for all strains originally preserved at the USDA- 5 39) were drawn from the Invertebrate Fungal Collection ARS Collection of Entomopathogenic Fungal Cultures and research team (Embrapa Genetic Resources and Biotech- Instituto de Patologia Tropical e Sau´de Pu´blica were nology, Brasilia DF, Brazil); the other isolates were from the included in the analyses. In addition, other Metarhizium USDA-ARS Collection of Entomopathogenic Fungal Cul- sequences from the same database were used as references tures (ARSEF 324, ARSEF 1914, ARSEF 1946, ARSEF 2024, in the phylogenetic tree (FIG. 1). Despite high consensus ARSEF 2575, ARSEF 3391) and Instituto de Patologia between GenBank and our own sequences for some cases in Tropical e Sau´de Pu´blica (Universidade Federal de Goia´s, which both were available for creation of phylogenetic trees, Brazil) (IP 30, IP 60, IP 119, IP 123, IP 143, IP 146) GenBank sequences were given priority over our own (TABLE I). With the exception of eight isolates (all six sequence data. ARSEF isolates plus CG374 and 645), all isolates had a Brazilian origin. These isolates were selected as being Creation of the main spectrum profile (MSP).—Conidial apparently representative of the Invertebrate Fungal Col- pellets of each of 11 reference strains were resuspended in lection in terms of genetic diversity and included insect 50 mL formic acid (70%), vortexed 1 min and exposed to a pathogens from orders Hemiptera, Coleoptera, Orthoptera, 15-min cycle in a bath-type ultrasonic washer (Unique, Lepidoptera and Hymenoptera or isolated from soil model USC-1800, Indaiatuba, Sa˜o Paulo, Brazil). Afterward, samples. Some strains were selected because they are active 50 mL acetonitrile (100%) were added to the mixture, ingredients of commercial mycoinsecticides or because they vortexed 1 min and sonicated for additional 15 min before currently are being used in some of our ongoing research centrifugation (16 100 3 g for 2 min). The supernatant was projects (Lopes et al. 2013a, b). Isolate CG1123, whose diluted with deionized water (1 : 10 v/v), and 1 mL genotype excludes this fungus from the so-called PARB supernatant was directly spotted onto a MSP96 steel target LOPES ET AL.: MALDI-TOF MS APPLIED TO METARHIZIUM 867 plate (Bruker Daltonics GmbH, Bremen, Germany) and the three solid media were assessed before acid digestion. allowed to air-dry for 15 min. A 1 mL droplet of the a-cyano- Briefly, conidial suspensions (20 mL) were pipetted onto 4-hydroxycinnamic acid (CHCA) matrix solution (previous- PDA and Petri dishes were sealed with parafilm after water ly prepared by dissolving 10 mg matrix powder [Bruker evaporation from suspension droplets and then incubated Daltonics GmbH] in 100 mL trifluoroacetic acid (TFA 3%) + at 25 6 0.5 C in darkness for 18 h. Each treatment was 400 mL deionized water + 500 mL acetonitrile) was applied repeated four times, and 300 conidia were scored in each over the dried sample and air-dried 15 min. Analyses were replicate at 4003 magnification. A conidium was consid- performed on a MicroFlex LRF MALDI-TOF mass spec- ered to have germinated if the germ tube was at least as long trometer (Bruker Daltonics GmbH) with a nitrogen laser as the length of an ungerminated conidium. (337 nm) with 20–65% intensity and operating in spiral acquisition mode. A protein extract of Escherichia coli Validation of MALDI-TOF MS for identification of (Bruker Daltonics GmbH) was used for external calibration Metarhizium species.—All 51 Metarhizium strains grown of this equipment. Each spectrum was obtained after an on PDA were analyzed by MALDI-TOF mass spectrometry. average of 240 laser shots (40 laser shots at six different spot Sample preparation was the same as described for pure positions) at 60 Hz, and signals were automatically collected conidia, and each strain was spotted in triplicates onto a with the AutoXecute tool of the FlexControl acquisition MSP96 steel target plate. Data were processed with Biotyper software (3.3; Bruker Daltonics GmbH) at a mass range of 3 software, and all peaks in the mass range 3000–15 000 m/z 2000–20 000 m/z under linear mode. Spectra were used in were considered. The collected spectra were processed with further analyses when the peaks showed a resolution higher baseline subtraction (multipolygon) and Savitsky-Golay than 200. Twenty-four independent samples (four conidial smoothing. The number of relevant peaks in mass spectra samples collected from six different cultures) were analyzed was limited to 100, and only peaks with a signal/noise ratio for each selected strain. Data were automatically exported . 3 were used. The spectra were analyzed with the standard to the Biotyper software (3.0; Bruker Daltonics GmbH) and pattern-matching algorithm to match the number and each consensus spectrum incorporated into the main intensity of peaks in the raw spectra with those in the spectrum profile (MSP) database. The consensus spectrum reference libraries. Results of the pattern-matching process consists of a profile created from the peaks retrieved from at of the tested strains spectra against the MSP database were least 20 spectra collected under the same conditions from expressed as log(score) values in a 0–3 scale, and those independent samples of any given isolate. The software , 1.70 (average for three independent samples) were rated performs all smoothing, normalization, baseline subtraction by us as ‘‘unidentified’’. In addition, spectra of all 51 strains and peak–picking operations, thereby creating a list of the were submitted to cluster analysis (MSP dendrogram most significant peaks (m/z values) for each spectrum and creation standard method 1.4) performed by Biotyper their corresponding intensities. Peak frequency in all software. This analysis calculates the distance between spectra for MSP creation was at least 25%. values by a correlation function and creates a dendrogram from the calculated distance values by a statistical algorithm Effect of conidial quantity on MALDI-TOF mass spectral in this manufacturer’s proprietary software. signals.—Different conidial amounts (2.0, 4.0 or 20 mg) from 10–12 d old cultures of three Metarhizium species (M. robertsii CG520; M. pingshaense CG1091, M. anisopliae RESULTS CG1125) were used in this preliminary experiment. Conidia from individual strains grown on PDA were carefully Molecular identification of Metarhizium strains.— harvested and processed as explained above. Each sample Bayesian and maximum likelihood phylogenetic was spotted onto a MSP96 steel target plate in triplicate analyses produced similar topologies, with Metarhi- prior to spectral acquisition. zium strains distributed in seven species within the M. anisopliae complex and one species outside this Effects of mycelial fragments and growth media for conidial production.—Mycelial fragments from 3-day-old cultures, a complex (TABLE I, FIG. 1). Species comprising the mixture of mycelium and conidia from 6-day-old cultures PARB (M. pingshaense-anisopliae-robertsii-brunneum) (ca. 1 : 1), or only conidia from 10-day-old cultures of the clade proposed by Bischoff et al. (2009) were the most same Metarhizium strains mentioned in the previous common: M. anisopliae sensu stricto (n 5 21), M. subsection, all grown on PDA, were subjected to MALDI- robertsii (n 5 8), M. pingshaense (n 5 1) and M. TOF MS analyses as previously described. M. anisopliae brunneum (n 5 1). M. anisopliae sensu stricto CG1125 also was grown on minimal medium (MM; 0.6% segregated into groups A and B, clearly clustering NaN03, 0.15% KH2PO4, 0.05% MgSO4?7H2O, 0.05% KCl, with ARSEF 6347 and the ex-type isolate, ARSEF 7487, 0.0001% FeSO , 0.0001% ZnSO ,1% glucose, 1.5% agar) or 4 4 respectively (FIG. 1). Our Group C (FIG. 1) comprised Sabouraud dextrose agar amended with yeast extract six strains (five comparatively similar and one more (SDAY; 1% glucose, 0.3% malt extract, 0.5% peptone, 0.3% yeast extract, 1.5% agar) to assess the effect of genetically divergent isolate) that are genetically close different media on the mass spectral signals. Conidia from to M. anisopliae sensu stricto but that could not be 10 d old cultures of M. anisopliae CG1125 were collected confidently assigned to that species. Likewise two and processed as described for MALDI-TOF MS analyses. other groups of isolates identifiable genotypically as Germination percentages of fresh conidia harvested from M. robertsii (groups D, E), clustered with strains 6 M 868

TABLE I. Metarhizium strains and their identifications by gene sequencing (TEF-1a region) and MALDI-TOF mass spectrometry

Molecular identification MALDI-TOF MS Strain codea GenBank code Origin Host or substrate Species Identity (%)b Species Scorec CG30 AY445082d Brazil Deois flavopicta (Hemipt.: Cercopidae) M. anisopliae 98.4 M. anisopliae 2.65 CG31 KF357940 Brazil Deois flavopicta (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.16 CG33 KF357931 Brazil Deois flavopicta (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.19 CG43 KF357932 Brazil Zulia entreriana (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.34 CG46 KF357936 Brazil Deois incompleta (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.31 CG47 KF357937 Brazil Galactica sp. (Lepidopt.: Galacticidae) M. anisopliae 99.4 M. anisopliae 2.30 CG53 KF357926 Brazil Deois flavopicta (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.21 CG55 KF357935 Brazil Deois flavopicta (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.11 CG57 KF357927 Brazil Deois flavopicta (Hemipt.: Cercopidae) M. anisopliae 99.3 M. anisopliae 2.20 CG144 KF357938 Brazil Piezodorus guildinii (Hemipt.: Pentatomidae) M. anisopliae 99.4 M. anisopliae 2.37 CG168 KF357939 Brazil Tibraca limbativentris (Hemipt.: Pentatomidae) M. anisopliae 99.4 M. anisopliae 2.52 CG491 KF357930 Brazil Deois sp. (Hemipt.: Cercopidae) M. anisopliae 99.4 M. anisopliae 2.59 CG1125e KF357921 Brazil Solenopsis invicta (Hymenopt.: Formicidae) M. anisopliae 99.3 M. anisopliae (2.54) CG1232 KC832295 Brazil Phyllophaga capillata (Coleopt.: Melolonthidae) M. anisopliae 99.4 M. anisopliae 2.27

CG1120e KC520539 Brazil soil M. anisopliae 100 M. anisopliae (2.54) YCOLOGIA CG1121 KF357925 Brazil soil M. anisopliae 100 M. anisopliae 2.37 CG1122 KC520540 Brazil soil M. anisopliae 99.8 M. anisopliae 2.32 CG1127 KF357916 Brazil Oebalus poecilus (Hemipt.: Pentatomidae) M. anisopliae 100 M. anisopliae 1.73 CG1235 KF357917 Brazil Glyphepomis spinosa (Hemipt.: Pentatomidae) M. anisopliae 100 M. anisopliae 1.92 CG1124 KC520542 Brazil soil M. anisopliae 100 M. anisopliae 2.42 IP 119 JQ061234d Brazil soil M. anisopliae 100 M. anisopliae 2.02 IP 30 JQ061190d Brazil soil unidentifiedf 98.7 unidentified 1.23 IP 60 JQ061182d Brazil soil unidentifiedf 98.8 unidentified 1.24 CG814 KF357928 Brazil Hedypathes betulinus (Coleopt.: Cerambycidae) unidentifiedf 98.8 unidentified 1.03 CG835 KF357929 Brazil Scaptocoris castanea (Hemipt.: Cydnidae) unidentifiedf 98.7 unidentified 1.12 CG1233 KC832296 Brazil Phyllophaga capillata (Coleopt.: Melolonthidae) unidentifiedf 99.0 unidentified 0.86 CG1231 KC832294 Brazil Phyllophaga capillata (Coleopt.: Melolonthidae) unidentifiedf 99.0 unidentified 1.03 CG1123 KC520541 Brazil soil unidentified 95.8–86.2g unidentified 1.23 CG210 KF357920 Brazil Cerotoma arcuata (Coleopt.: Chrysomelidae) M. robertsii 99.7 M. robertsii 2.28 CG520e KF357913 Brazil Stiphra robusta (Orthopt.: Proscopiidae) M. robertsii 99.7 M. robertsii (2.44) CG1234 KC832297 Brazil Phyllophaga capillata (Coleopt.: Melolonthidae) M. robertsii 99.7 M. robertsii 2.02 CG632e KF357914 Brazil Acromyrmex sp. (Hymenopt.: Formicidae) M. robertsii 100 M. robertsii (2.10) ARSEF 2575 KC484650 USA Curculio caryae (Coleopt.: Curculionidae) M. robertsii 100 M. robertsii 1.81 IP 123 JQ061240d Brazil soil M. robertsii 99.7 M. robertsii 1.98 IP 146 JQ061236d Brazil soil M. robertsii 99.7 M. robertsii 2.11 CG1224 KF357923 Brazil soil M. robertsii 100 unidentified 1.53 CG374e KF357924 Philippines (Coleopt.: Scarabaeidae) M. majus 100 M. majus (2.09) TABLE I. Continued

Molecular identification MALDI-TOF MS

Strain codea GenBank code Origin Host or substrate Species Identity (%)b Species Scorec

CG27 KF357915 Brazil unknown M. majus 100 M. majus 1.92 L ARSEF 1914 EU248868d Philippines Oryctes rhinoceros (Coleopt.: Scarabaeidae) M. majus 100 M. majus 2.10 AL ET OPES ARSEF 1946 EU248867d Philippines Oryctes rhinoceros (Coleopt.: Scarabaeidae) M. majus 100 M. majus 2.25 CG423e KF357918 Brazil Schistocerca pallens (Orthopt.: Acrididae) M. acridum 100 M. acridum (2.13) CG288 KF357919 Brazil Schistocerca pallens (Orthopt.: Acrididae) M. acridum 100 M. acridum 1.84 ARSEF 324 EU248844d Australia Austracris guttulosa (Orthopt.: Acrididae) M. acridum 99.4 M. acridum 1.89 MS MALDI-TOF .: ARSEF 3391 EU248873d Tasmania Zonocerus elegans (Orthopt.: Acrididae) M. acridum 99.6 M. acridum 1.83 CG1091e KC520538 Brazil Cosmopolites sordidus (Coleopt.: Curculionidae) M. pingshaense 99.5 M. pingshaense (2.41) CG1126e KF357922 Brazil Eurhizococcus brasiliensis (Hemipt.: Margarodidae) M. brunneum 98.9 M. brunneum (2.42) CG1237 KF357933 Brazil Aegopsis balboceridus (Coleopt.: Melolonthidae) M. lepidiotae 96.2 M. lepidiotae 2.21 CG1238e KC832298 Brazil Aegopsis balboceridus (Coleopt.: Melolonthidae) M. lepidiotae 96.2 M. lepidiotae (2.07) ARSEF 2024e DQ463965d France Otiorhynchus sulcatus (Coleopt.: Curculionidae) M. flavoviride 100 M. flavoviride (2.30) var. flavoviride var. flavoviride PLE TO APPLIED CG645e KF357934 France Pemphigus trehernei (Hemipt.: Pemphigidae) M. flavoviride 100 M. flavoviride (2.38) var. pemphigi var. pemphigi IP 143 JQ061180d Brazil soil M. flavoviride 98.1 M. flavoviride 1.88 var. pemphigi var. pemphigi M a Isolates with codes CG, ARSEF, and IP were drawn from the Invertebrate Fungal Collection at Embrapa Genetic Resources and Biotechnology (Brasilia, Federal ETARHIZIUM District, Brazil), USDA-ARS Collection of Entomopathogenic Fungal Cultures (Ithaca, New York) and Instituto de Patologia Tropical e Sau´de Pu´blica (Universidade Federal de Goia´s, Goiaˆnia, Brazil) respectively. b Similarity of TEF-1a sequences with ARSEF reference strains (ex-type or ex-epitype) isolates. c Score produced by the comparison of a spectrum against the MSP database (# 1.69 5 unidentified; $ 1.70 5 species/variety identification). A number in parentheses refers to a score obtained when that strain used to construct the MSP database was compared against itself. d DNA sequences retrieved from GenBank. e Selected strains used to construct the MSP databases. f Group with strains close to M. anisopliae sensu stricto according to Rocha et al. (2012). g Range refers to homology with all ex-type or ex-epitype isolates used in this study. 869 870 MYCOLOGIA LOPES ET AL.: MALDI-TOF MS APPLIED TO METARHIZIUM 871

FIG. 2. Comparative differences among matrix-assisted laser desorption ionization-time-of-flight (MALDI–TOF) mass spectra for different Metarhizium species.

ARSEF 6472 and the ex-type isolate, ARSEF 2575, M. flavoviride var. flavoviride (n 5 1) and M. respectively. Outside the PARB clade, M. majus (n 5 flavoviride var. pemphigi (n 5 2) also were identified, 4), M. lepidiotae (n 5 2), and M. acridum (n 5 4) clustering with strains ARSEF 2133 and ARSEF 7491 isolates clustered with strains ARSEF 1858, ARSEF respectively. Isolate CG1123, with a genotype from 4154 and ARSEF 7486 respectively. In addition to outside the PARB clade (Lopes et al. 2013b) and used these representatives of the M. anisopliae complex, here as a further control for MALDI-TOF profiles, r

FIG. 1. Bayesian majority rule consensus phylogram of aligned 59 end of TEF-1a gene sequence data for Metarhizium strains. Support values were given as the Bayesian prior probability (first number) and percentage of bootstrap support derived from a MP analysis (second number). Isolates marked with a T are ex-type; those marked with an asterisk were used to construct the main spectrum profiles. 872 MYCOLOGIA

FIG. 3. Dendrogram showing cluster analysis of MALDI-TOF mass spectra. Isolates marked with an asterisk were used to construct the main spectrum profiles. returned a protein profile that, as should be TOF signal quality for the recognition of M. robertsii expected, did not match any known species in our CG520, M. pingshaense CG1091 or M. anisopliae study. CG1125 in that log(score) values were $ 2.10 (SUPPLEMENTARY FIG. 1). On the other hand, mycelial Creation of the main spectrum profile (MSP).—Based samples produced low quality spectra and interfered on the phylogenetic analysis, nine strains with high with identification of all three strains, with score homology to corresponding ex-type or ex-epitype values in the 0.24–0.84 range, values that were strains initially were selected for reference spectrum considerably below the cut-off limit of 1.70 (SUPPLE- acquisition aiming creation of reference libraries. MENTARY FIG. 2). Although the use of mixed samples These isolates were M. anisopliae CG1125 (Group B; (mycelial fragments + conidia) was not problematic see FIG. 1 or TABLE I), M. robertsii CG520 (Group D), for M. robertsii CG520 or M. pingshaense CG1091, the M. pingshaense CG1091, M. brunneum CG1126, M. log (score) value for the mixed sample was only 1.56 majus CG374, M. lepidiotae CG1238, M. acridum for M. anisopliae CG1125, whereas score values of this CG423, M. flavoviride var. flavoviride ARSEF 2024, isolate obtained from only conidia were always $ 2.39. and M. flavoviride var. pemphigi CG645. The raw Last, the initial viability of conidia harvested from spectra of these nine species adopted in the first cultures of M. anisopliae on SDAY, PDA and MM and reference library are illustrated (FIG. 2). A second used in the MALDI-TOF MS analyses were 99.5%, reference library also was built to include represen- 99.2% and 63.5% respectively. Spectra obtained from tation of all clearly identified taxa and groups by M. anisopliae CG1125 conidia produced on SDAY and adding strains from groups A (M. anisopliae sensu PDA matched the reference library, with scores of stricto CG1120) and E (M. robertsii CG632). 2.13 and 2.20 respectively. Growth on MM produced a slightly different profile and a much lower identifica- Influences of conidial quantity, culture media and tion score of 1.39 (SUPPLEMENTARY FIG. 3). fungal cell types on MALDI-TOF MS mass spectral signals.—The quantity of conidia used in the Validation of MALDI-TOF MS for identification of hydrolysis process did not interfere with MALDI- Metarhizium species.—When the original 9 strain LOPES ET AL.: MALDI-TOF MS APPLIED TO METARHIZIUM 873 reference library was used, no strains from M. nomic groups among these MALDI-TOF profiles anisopliae sensu stricto and M. robertsii groups A differed from those in their gene sequence-based and E (groups not containing isolates with high analyses (e.g. FIG. 1, and Bischoff et al. 2009). genetic homology to the respective ex-type or ex- epitype strains) could be reliably identified because DISCUSSION their average log(scores) were considerably below the cut-off value of 1.70. However, when we adopted the Fungal identification by MALDI-TOF mass spectrom- more comprehensive library–the original nine isolates etry is based on comparisons of protein spectrum plus one additional reference strain from both profiles of unidentified strains against the reference Groups A and E–accurate MALDI-TOF identifications profiles of known taxa. While it is inherently a were obtained for all 19 non-reference isolates of M. molecular technique, MALDI-TOF differs significant- anisopliae sensu stricto and for five of the six non- ly from the direct comparisons of gene sequences reference M. robertsii strains tested. used for phylogenetically based systematics. Despite In addition to the 11 strains in the expanded the universal dependence on sequence-based system- reference library, MALDI-TOF profiles were generat- atics as a methodological approach for revising fungal ed for 40 additional test strains of Metarhizium. systematics and discovering phylogenetic relation- Robust matches with the reference strains (with score ships, it is useful to remember that MALDI-TOF values $ 1.70) were obtained for 39 out of 40 test profiles show differences among large numbers of strains. M. robertsii CG1224 was the only strain whose unidentified proteins reflecting a much larger pro- genomically determined identity was not unambigu- portion of a total organismal genome than is now ously confirmed by mass spectrometry despite repeat- routinely used with multigenic sequence-based stud- ed analyses of this isolate. Despite its relatively low ies. These two dramatically different approaches score against the M. robertsii reference isolate, the MS should be accepted as useful in their own contexts. profile of CG1224 is more similar to the profiles of M. In this study, we created main spectrum profiles for robertsii than to any other taxon tested here. As might reference strains (those with high genetic homology be expected, the seven isolates whose genetic identi- to ex-type and ex-epitype isolates) that allowed fications were not available all yielded inconclusively accurate MALDI-TOF identifications (agreeing with low log(scores) 0.86–1.24. Because only single strains identifications based on the TEF-1a gene fragment) of M. pingshaense, M. brunneum and M. flavoviride of 39 out of 40 non-reference strains from a number var. flavoviride were available for the MALDI-TOF of species within the Metarhizium anisopliae complex. study, these species were still incorporated into the We do not believe and do not wish to imply that MS- reference libraries but the effectiveness of this based identification of fungi can or should replace technique for diagnosing these species has not been their gene-based identifications that have become the adequately tested; the addition of further strains will de facto required standard for almost all taxonomic certainly improve the reliability of the MALDI-TOF studies. approach for identifying these taxa. Further, a group The robustness of the MALDI-TOF MS approach of genetically similar Brazilian strains (IP 30, IP 60, for Metarhizium species identification is highly influ- CG814, CG835, CG1231, CG1233) that are also enced by both sample preparation procedures (as genetically similar to but not verifiably identical with shown here) and by diverse sorts of contaminants M. anisopliae sensu stricto proved to have protein affecting mass spectrometers (Keller et al. 2008). We spectra forming their own distinct cluster, labeled adjusted a protein extraction method from aerial here as Group C (FIG. 3). The taxonomic status of conidia, but the presence of hyphae (mycelial these isolates clearly deserves further evaluation with fragments) in the test preparation can influence the multigenic phylogenetic studies. The lack of MS- spectral signal and, therefore, its matching against the based correspondence of strain CG1123 with any standard reference profiles. These results confirmed other species used here similarly underscores the the importance of standardizing growth conditions need for further gene-based characterization of this and other methodologies before MALDI-TOF analy- strain. ses and also underscore previous studies that proteins The dendrograms of protein-based MALDI-TOF are differentially expressed by conidia and mycelium, MS identification profiles closely matched those of as was shown for the protein-expression profiles of M. the gene-based phylograms from Bischoff et al. acridum mycelium and conidia in which only 35% of (2009). This mass spectrometric approach successful- the proteins were common to both cell types (Barros ly recovered groupings of taxa that are essentially et al. 2010). Studies of filamentous fungi by MALDI- identical to those from genomic analyses. Nonethe- TOF MS based on vegetative cells usually yielded less, the relative distances and placements of taxo- satisfactory identifications for species of Trichoderma 874 MYCOLOGIA

(de Respinis et al. 2010); these authors concluded highly confident MS-based identifications, these that MALDI-TOF MS analyses of 2 d old mycelial strains clearly grouped with the ex-type isolate, ARSEF samples were useful for species delimitation in this 7486, in the phylogenetic analysis, all were obtained genus. Similarly Chowdappa et al. (2013) showed from acridid hosts and their morphological features significant MALDI-TOF capacity to identify Alternaria (e.g. their conidial dimensions and pale colony color) species by analyzing intact 5 d old mycelium. also were consistent with their traditional gene-based However, we observed both higher quality and better identification as M. acridum. Therefore, the lower cut- reproducibility for protein spectra using only conidia off value adopted in our study (1.70) was supported by of Metarhizium. Nonetheless, conidia produced on a morphological traits. This integration of morpholog- low-nutrient medium (MM) showed both different ical, genetic sequence and protein profiling approach- protein profiles and poorer germination than those es has been referred to as an advantageous strategy for from high-nutrient media (PDA or SDAY). Although the reliable identification of fungi (Wicht et al. 2012). conidia formed on nutritionally marginal substrates A notable exception to these criteria for identifica- may not be so completely developed as those on tion was isolate CG1224 (M. robertsii Group E), which nutritionally richer substrates, the more important showed 100% genetic homology to its corresponding observation for Metarhizium species is that the most ex-type isolate but a MALDI-TOF score of only 1.53. informative proteins for compiling MALDI-TOF CG1224 was the only example we encountered of a profiles were obtained from conidia only, rather than strain that could not be spectrometrically matched from mycelium or mycelial/conidial mixtures. with its genotypically identified reference. Nonethe- This study highlighted the importance of using the less, its MS profile was closer to the M. robertsii strains broadest possible selection of representative strains to used to construct the MSP database than to any other create reference libraries. Libraries based only on ex- tested taxon. Since our expectation in this study was type or ex-epitypes isolates (or, as was our case, on to achieve matching of identifications between MS isolates showing high genetic homology to these and the current genetically based classification taxonomic standard isolates) did not support un- system, we could have chosen a lower cut-off value equivocal identifications of a number of unknown (e.g. #1.53), but the adoption of a more stringent strains. However, when we added several more isolates limit gave us the confidence that all other MS of M. anisopliae sensu stricto and M. robertsii isolates identifications were correct and also provided stron- to our MS-based molecular studies, we were able to ger clues about which isolates need further se- distinguish clearly two subgroups within each of these quenced-based taxonomic evaluation. two species. By adding representatives of these two Among the seven strains that were tested without groups not previously considered (CG1125 and genomically confirmed identifications, six of them, CG520 of M. anisopliae sensu stricto Group A and including isolates IP 30 and IP 60 (Rocha et al. 2012) M. robertsii Group E respectively), non-reference and CG1231 and CG1233 (Lopes et al. 2013a), belong strains from these taxa used in this work could be to a sister group related to M. anisopliae (s.l.), robustly identified by spectrometric analyses. In clustered together and are designated here as Group general, a high degree of gene sequence similarities C. According to these studies, additional molecular between unknown isolates and their corresponding characterization will be required to determine wheth- ex-type/ex-epitypes was correlated with MALDI-TOF er these isolates might represent a new taxon. Indeed, MS scores $ 2.00 (a default value commonly used in our Group C isolates appear to be similar to another similar studies). Exceptions found here were the group of Brazilian isolates that is already being more three M. acridum strains, with genetic homology in intensively studied with a multigenic sequence ap- the 99.4–100% range but that had MALDI-TOF MS proach (S. Rehner pers comm). No isolate from this scores in the 1.83–1.89 range, and with five other group was added to the main reference library seemingly anomalous isolates. Both CG1127 and because those libraries were specifically intended to CG1235 (M. anisopliae Group A; see FIG. 1) showed include only fungi identified definitively by genomic 100% genetic homology but MALDI-TOF scores with characters. We think that Group C probably repre- their correct group of only 1.73 and 1.92 respectively. sents an undescribed cryptic species of Metarhizium IP 123 (M. robertsii Group E) showed 99.7% genetic but would not include any isolate from this group in homology but a MALDI-TOF score of 1.98; CG27 (M. the reference library until more complete gene-based majus) showed 100% genetic homology with its ex- studies can confirm this probable status. If any Group type but a score of 1.92; and IP 143 (M. flavoviride var. C isolate were placed into the main reference library, pemphigi) showed 98.1% genetic homology but a all remaining isolates belonging to its group would be score of 1.88. Although MALDI-TOF MS scores for M. retrieved with significantly high score values as acridum were slightly below the default value for identifiable with that reference isolate. LOPES ET AL.: MALDI-TOF MS APPLIED TO METARHIZIUM 875

TABLE II. Comparative estimates of time and reagent costs between single-gene phylogeny and mass spectrometric analyses (MALDI-TOF) for each group of 100 isolates within the Metarhizium anisopliae complex

Processing step DNA sequencing MALDI-TOF

Initial growth and processing Cultivation of fungus on solid medium 10–12 d 10–12 d Extraction of proteins from conidia — 2 d Protein processing — , 6h Cultivation of fungi in liquid medium 3 d —a First analysis Mycelial processing 2–3 d —a DNA extraction 2–3 d —a PCR amplification 1–2 h —a Sequencing/purification or MALDI-TOFb 3–4 h 2–3 h Data analyses 1–3 d , 1h Estimated total time for processing 18–24 d 12–14 d Estimated cost for reagentsb US$ 515.00 US$ 2.00 Re-analysis to accommodate new phylogenetic same as above only for reference isolates reclassification from new taxac

a For the MALDI-TOF approach, gene sequencing is usually unnecessary, unless in the rare occasions in which one or more reference isolates used to compile the Main System Profile library have not been sequenced before (as was the case in the present study). b Contrary to the sequencing approach, only reference isolates from new taxon/taxa have to be subjected to MALDI-TOF when a new phylogenetic classification is proposed, followed by data analyses with all isolates (including those protein profiles previously stored). c Estimated cost in US$ for analyzing 100 isolates at the Invertebrate Fungal Collection (Brasilia, Brazil) considering steps following conidial/mycelial processing. Estimates for reagents were based on concentrations adopted in this publication. Additional costs, such as labor (1–2 people) and equipment (acquisition/maintenance), were not included. Based on our experience, commercial services of sequencing/purification would cost ca. US$1200.00 per 100 isolates.

Another strain, CG1123, isolated from soil in a degree of phylogenetically determined relatedness of banana plantation, was known to be within the M. the sister groups M. anisopliae (s.str.) and M. robertsii anisopliae complex but not within the PARB clade (Bischoff et al. 2009) was not mirrored by MALDI- (Lopes et al. 2013b). This fungus was not unambig- TOF analyses; these taxa are well distinguished by uously identifiable by either gene sequencing or their MALDI-TOF profiles but were placed relatively spectrometric analyses and needs further multiple distant from each other on the MS-based dendrogram gene-based evaluation as a possible new taxon. (FIG. 3). Indeed, M. anisopliae groups A and B were Further resolution of the genus Metarhizium outside separated by a M. pingshaense strain (CG1091) the PARB clade (Kepler et al. 2014) also is required to whereas M. robertsii groups D and E clustered unravel both the phylogeny and specific identifica- together but at the opposite extreme of the dendro- tions of this fungus; once the corresponding main gram and, therefore, in conflict with the grouping of mass spectrometric protein profiles are generated for the PARB clade seen in gene-based phylogenetic trees Metarhizium species outside the M. anisopliae species (FIG. 1; also see Bischoff et al. 2009). It is likely that complex, MALDI-TOF should become an even more the use of more specifically targeted and larger valuable tool to flag individual isolates that do not fit numbers of molecular markers in addition to those well within the current taxonomy of this genus and now commonly used for fungal phylogenetics will are candidates for more complete characterization better define variability within the M. anisopliae and possible description as new cryptic taxa. complex, as was recently shown by Kepler and Rehner While MALDI-TOF MS and gene sequencing (2013). Note, however, that MALDI-TOF confirms the resulted in highly similar groupings within Metarhi- assignments of isolates to each of these groups and zium, spectrometry did not perfectly reflect gene that the strength of this spectrometric technique is in sequence-based phylogeny (Bischoff et al. 2009); a providing basic specific identification with a high similar result also was observed for some Trichoderma degree of confidence. The degree of phylogenetically clades by de Respinis et al. (2010) and for Alternaria determined relatedness between these groups species by Brun et al. (2013). The apparently close (whether or not they represent discrete taxa) is an 876 MYCOLOGIA appropriate question to ask of sequence-based studies MALDI-TOF MS for preliminary species identifica- but is emphatically not a relevant issue for the use of tions with a high degree of confidence for entomo- MALDI-TOF mass spectrometry. The utility of pathogenic (and, eventually, other) fungi is a major MALDI-TOF is limited to taxa for which good accomplishment. In spectrometric analyses, strains reference libraries of verifiably identified and gener- would need to be analyzed only once and, whenever ally closely related (congeneric) isolates are compiled; necessary, taxonomic updating of an entire collection it cannot serve adequately to identify random, wholly after phylogenetic reclassifications is easily and unidentified isolates. There is no overall reference rapidly accomplished by comparing stored profiles database of MALDI-TOF profiles comparable to with those of newly proposed taxa. In contrast, the GenBank or other genomic databases against which addition of one or more new gene sequences in any BLAST queries with a single gene sequence may allow genomically based taxonomic revision nominally more or less reasonably meaningful identifications. would require the longer, more expensive, and more The relatively low operational cost and rapidity of complex task of sequencing of all affected isolates for the spectrometric approach in comparison to gene the new gene(s) to verify or to adjust their identifi- sequencing deserves careful evaluation in that PCR cations. The satisfactory degree of taxonomic accura- and sequencing (including commercial services) are cy reported here is vital when recommending this more or less globally ubiquitous and available at ever- comparatively rapid approach as an alternative to decreasing costs. We must recognize first, however, gene sequencing for preliminary screening of fungal that the capital expense for either DNA sequencers or biodiversity, ecology and population biology that, by the mass spectrometric equipment for MALDI-TOF their very nature, often tend to involve dauntingly analyses is prohibitively high for most individual large numbers of isolates. laboratories. These methodological approaches may be most practical if individual scientists are able to send their material to properly equipped and ACKNOWLEDGMENTS maintained central facilities; this practice is already We thank Drs Stephen Rehner and Ryan Kepler (USDA- common for obtaining DNA sequences. Compelling ARS, Beltsville, Maryland) for their kind cooperation in evidence (TABLE II) reveals that, apart from the high providing supplemental information and sequence data. costs of the hardware for either sequence-based or MS studies, MALDI-TOF approaches indeed are both rapid and inexpensive in a point-by-point comparison LITERATURE CITED of the time and costs for on-demand processing of Anhalt JP, Fenselau C. 1975. Identification of bacteria using isolates by PCR/gene sequencing and MALDI-TOF mass spectrometry. Anal Chem 47:443,219–225, techniques. Keeping in mind that the overall goals doi:10.1021/ac60352a007 may be quite different in choosing to use one over the Bader O, Weig M, Taverne-Ghadwal L, Lugert R, Grob U, other of these two approaches, there can be little Kuhns M. 2011. 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